METHOD FOR DIFFERENTIATING PLURIPOTENT STEM CELLS INTO DOPAMINERGIC NERVE CELLS IN MIDBRAIN SUBSTANTIA NIGRA
20230340407 · 2023-10-26
Assignee
Inventors
Cpc classification
C12N2501/119
CHEMISTRY; METALLURGY
International classification
Abstract
Specific method for differentiating pluripotent stem cells into dopaminergic (A9 mDA) nerve cells in the midbrain substantia nigra is provided. Mature A9 mDA neurons are formed by differentiation, which can express the molecular markers of the midbrain substantia nigra dopaminergic neurons, including TH, FOXA2, EN1, LMX1A, NURR1 and GIRK2, but which rarely express the marker CALB of the ventral tegmental area dopaminergic neurons. A9 mDA nerve cells are transplanted into the substantia nigra, and the axons can project to the target brain area which is innervated by endogenous substantia nigra dopaminergic neurons, the dorsal striatum; the transplanted A9 mDA neurons themselves exhibit the classic electrophysiological characteristics of endogenous substantia nigra dopaminergic neurons, including a low-frequency spontaneous discharge frequency, and can induce sag by means of hyperpolarizing current stimulation, and transplanting the A9 mDA nerve cells into the substantia nigra or striatum of individuals with neurodegenerative diseases can alleviate motor deficits.
Claims
1. A method for preparing midbrain substantia nigra dopaminergic neurons, comprising: (1) culturing stem cells in a medium containing neural induction agents, ie. supplementing components in consecutive multiple stages to accomplish induction; and (2) obtaining the stem cell-derived midbrain substantia nigra dopaminergic neurons from the culture.
2. The method according to claim 1, wherein in (1), said multiple stages for induction with supplementary components are: first stage: Adding SB431542, DMH-1, SHH and CHIR99021; second stage: adding SAG, SHH and CHIR99021; third stage: adding SHH, SAG and FGF8b; fourth stage: adding SHH and FGF8b.
3. The method according to claim 1, wherein, said multiple stages for induction with supplementary components are: first stage: adding 1~15 .Math.M DMH-1, 200~1000 ng/mL SHH, 0.1~1 .Math.M CHIR99021; second stage: adding 0.1 ~ 5 .Math.M SAG, 50-300 ng/ml SHH, 0.1 ~ 1 .Math.M CHIR99021; third stage: adding 5~100 ng/ml SHH, 0.1~5 .Math.M SAG, 5~200 ng/ml FGF8b; fourth stage: adding 5~100 ng/ml SHH, 5~80 ng/ml FGF8b.
4. The method according to claim 3, wherein, said multiple stages for induction with supplementary components are: first stage: adding 10±5 .Math.M SB431542, 2±1 .Math.M DMH-1, 500±200 ng/ml SHH, 0.4±0.2 .Math.M CHIR99021; second stage: adding 2±1 .Math.M SAG, 100±50 ng/ml SHH, 0.4±0.2 .Math.M CHIR99021; third stage: adding 20±10 ng/ml SHH, 0.5±0.2 .Math.M SAG, 100±50 ng/ml FGF8b; fourth stage: adding 20±10 ng/ml SHH, 20±10 ng/ml FGF8b.
5. The method according to claim 1, wherein, in said multiple stages: first stage: culturing for 6~8 days from the beginning; preferably 7±0.5 days; second stage: culturing for 6~8 days to 11~13 days; preferably 12±0.5 days; third stage: culturing for 11~13 days to 18~20 days; preferably 19±0.5 days; fourth stage: culturing for 18~20 days to 31-33 days; preferably 32±0.5 days.
6. The method according to claim 1, wherein the stem cells comprise: embryonic stem cells or induced pluripotent stem cells; preferably, the stem cells are human stem cells, the embryonic stem cells or induced pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells.
7. A midbrain substantia nigra dopaminergic nerve cell, wherein it is prepared by the method of claim 1.
8. A midbrain substantia nigra dopaminergic nerve cell, wherein it expresses molecular markers of midbrain substantia nigra dopaminergic neurons after differentiation for 5~10 days, said molecular markers comprising tyrosine hydroxylase, FOXA2, EN1, LMX1A, NURR1 and/or GIRK2; while it rarely expresses the marker CALB of the ventral tegmental area dopaminergic neurons.
9. The midbrain substantia nigra dopaminergic nerve cell according to claim 7, wherein, after the midbrain substantia nigra dopaminergic nerve cell being transplanted into the substantia nigra of brain and differentiation, A9 mDA neurons are obtained the axons thereof can specifically project to the target brain area-dorsal striatum which is innervated by endogenous substantia nigra dopaminergic neurons; and/ or the obtained A9 mDA neurons themselves exhibit the classic electrophysiological characteristics of the endogenous substantia nigra dopaminergic neurons, comprising a low-frequency spontaneous discharge frequency, and can induce sag by means of hyperpolarizing current stimulation; and/ or the obtained A9 mDA neurons in the substantia nigra or striatum of brains can alleviate motor deficits.
10. The midbrain substantia nigra dopaminergic nerve cell according to claim 7, wherein, the midbrain substantia nigra dopaminergic nerve cell is A9 mDA nerve cell; preferably, more than 80% of the cells express A9 mDA marker GIRK2 after differentiation for 5~10 days; more preferably, more than 85% of the cells express A9 mDA marker GIRK2.
11. The midbrain substantia nigra dopaminergic nerve cell according to claim 7, wherein, after differentiation for 5~10 days, more than 40% of total cells or more than 50% of the TUJ1+ neurons exhibit the characteristics of molecular markers; more preferably, more than 50% of total cells or more than 60% of the TUJ1+ neurons exhibit the characteristics of molecular markers.
12. A midbrain substantia nigra dopaminergic neuron obtained from the differentiation of the midbrain substantia nigra dopaminergic nerve cell of claim 7; preferably, it expresses molecular markers of midbrain substantia nigra dopaminergic neurons, comprising TH, FOXA2, EN1, LMX1A, NURR1 and/or or GIRK2, while it rarely expresses the marker CALB of the ventral tegmental area dopaminergic neurons.
13. A method for treating neurodegenerative diseases, comprising administrating the midbrain substantia nigra dopaminergic nerve cell according to claim 7 to required subjects.
14. A preparation for treating neurodegenerative diseases, wherein, it comprises: the midbrain substantia nigra dopaminergic nerve cell according to claim 7; and pharmaceutically acceptable carriers.
15. The method according to claim 13, wherein, the midbrain substantia nigra dopaminergic nerve cell is used as a graft for transplantation into the substantia nigra or striatum of the brain .
16. The method according to claim 13, wherein, the neurodegenerative diseases comprise: Parkinson’s disease, Alzheimer’s disease, Lewy body dementia, Huntington’s disease, amyotrophic lateral sclerosis, nerve damage.
17. A method for screening substances for improving neurodegenerative diseases, wherein the method comprises: (1) treating a model system by a candidate substance, wherein the model system is neural circuit damaged or neural function damaged which comprises midbrain dopaminergic neurons; and (2) evaluating, if the candidate substance can statistically promote dopaminergic neurons to repair damaged neural circuits in the brain or promote their remodeling of neural functions, then the candidate substance is a useful substance for repairing damaged neural circuits or reconstructing neural functions.
18. The method according to claim 17, wherein, the model system in step (1) is an animal model system, a tissue model system, an organ model system, a cell model system.
19. The method according to claim 17, wherein, step (2) comprises: observing the influence of the candidate substance to midbrain dopaminergic neurons, if it promotes midbrain dopaminergic neurons to repair nigra-striatal pathway, then it is a useful substance for repairing damaged neural circuits or reconstructing neural functions; or observing the influence of the candidate substance to midbrain dopaminergic neurons, if it promotes pre- and post-synaptic integration of midbrain dopaminergic neurons, then it is a useful substance for repairing damaged neural circuits or reconstructing neural functions; or observing the influence of the candidate substance to midbrain dopaminergic neurons, if it promotes the projection of axons of midbrain dopaminergic neurons to the dorsal region of striatum, then it is a useful substance for repairing damaged neural circuits or reconstructing neural functions; or observing the influence of the candidate substance to midbrain dopaminergic neurons, if it promotes the neural fiber formation of midbrain dopaminergic neurons, along with the endogenous nigra-striatal neural connected pathway, specific growth and extension to its endogenous target area-striatum to form neural connections with striatal neurons, and projection to the striatum, then it is a useful substance for repairing damaged neural circuits or reconstructing neural functions.
20. The method according to claim 17, wherein, the model system is an animal system, the animal has motor deficits, wherein the method also comprises: evaluating the motor abilities of animals, if the candidate substance can alleviate motor deficits, then it is a useful substance for repairing damaged neural circuits or reconstructing neural functions.
Description
BRIEF DESCRIPTION OF FIGURES
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DETAILED DESCRIPTION
[0156] The inventors have deeply studied and revealed a specific method for differentiating pluripotent stem cells into midbrain substantia nigradopaminergic nerve cells. The differentiated mature A9 mDA neurons can express the molecular markers of the midbrain substantia nigra dopaminergic neurons, including TH, FOXA2, EN1, LMX1A, NURR1 and GIRK2, while rarely express the marker CALB of the ventral tegmental area dopaminergic neurons . The A9 mDA nerve cells can be transplanted into the substantia nigra, and the axons thereof can specifically project to the target brain area-dorsal striatum which is innervated by endogenous substantia nigra dopaminergic neurons; the grafted A9 mDA neurons receive more inhibitory but less excitatory inputs, whose regulation is similar to that of the endogenous substantia nigra dopaminergic neurons; the transplanted A9 mDA neurons themselves exhibit classic electrophysiological characteristics of the endogenous substantia nigra dopaminergic neurons, including a low-frequency spontaneous discharge frequency, and can induce sag by means of hyperpolarizing current stimulation; transplanting the A9 mDA nerve cells into the substantia nigra or striatum of individuals with neurodegenerative diseases can alleviate motor deficits.
Terms
[0157] As used herein, the term “treatment” or “treat” here includes preventive (e.g., prophylactic), curative, or palliative treatment of a mammal, especially human; and includes (1) preventing, treating, or alleviating a disease (such as cancer) in an individual, and the individual is at high risk of developing the disease, or has the disease but has not yet been diagnosed; (2) inhibiting a disease (eg, inhibiting its occurrence); or (3) alleviating a disease (eg, alleviating symptoms associated with the disease).
[0158] As used herein, the “midbrain substantia nigra dopaminergic nerve cells”, “stem cell-derived midbrain substantia nigra dopaminergic nerve cells” and “A9 mDA nerve cells” may be used/ referenced interchangeably.
[0159] As used herein, the “cells” comprises “a population of cells” and also comprises “cell cultures”.
[0160] As used herein, “individual”, “patient”, “participant” or “subject” refers to animals (such as rodents, primates) including humans, which can receive treatments of cells in the present disclosure (midbrain substantia nigra dopaminergic nerve cells) or cell preparations.
[0161] As used herein, “prevention” comprises “prevention”, “alleviation” and “treatment”.
[0162] As used herein, “neural circuit” refers to connected neurons with different properties and functions in brain through various forms; in the present disclosure, the nigra-striatal neural circuit is particularly concerned.
[0163] As used herein, “pharmaceutically acceptable” ingredients refers to substances suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity), ie. with reasonable benefit/risk ratio. The term “pharmaceutically acceptable carrier” refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents. The term refers to a pharmaceutical carrier that is, by itself, not essential active ingredients and is not unduly toxic after administration.
[0164] As used herein, “effective amount” refers to the amount of agents (cells or cell preparations in the present disclosure) that are sufficient to obtain desired curative effects. Effective amounts also include instances where the therapeutically beneficial effects of the agents outweigh toxic or detrimental effects thereof. Effective amount of agents does not necessarily cure diseases or disorders, but can delay, hinder or prevent the occurrence of the diseases or disorders, or alleviate the symptoms associated with the diseases or disorders. A therapeutically effective amount may be divided into one, two or more doses and administered once, two or more times in an appropriate dosage form within a given period.
Stem Cell-Derived Neurons and Its Preparation
[0165] The present disclosure provided midbrain substantia nigra dopaminergic nerve cells (or cell populations), and they are mainly A9 mDA nerve cells.
[0166] As an embodiment of the present disclosure, in stem cell-derived neurons obtained by the present disclosure, more than 80% express the A9 mDA neurons marker GIRK2; more preferably, more than 85% express the A9 mDA neurons marker GIRK2.
[0167] As an embodiment of the present disclosure, the midbrain substantia nigra dopaminergic neurons express markers of FOXA2, LMX1A and EN1. If further differentiated for about a week, they also express tyrosine hydroxylase (TH) and EN1, FOXA2, LMX1A and NURR1 (preferably more than 40% of total cells or more than 50% of cells in TUJ1+ neurons exhibit said characteristics; more preferably, more than 50% of the total cells or more than 60% of the cells in TUJ1+ neurons exhibit said characteristics).
[0168] The present disclosure also provided a method for preparing midbrain substantia nigra dopaminergic nerve cells in vitro, including: (1) culturing stem cells in a medium containing neural induction agents; and (2) obtaining the midbrain substantia nigra dopaminergic nerve cells from the culture.
[0169] As an embodiment of the present disclosure, in (1), adding neural induction agents into the medium; and inducing separately in multiple stages with supplementary components: first stage: adding SB431542, DMH-1, SHH and CHIR99021; second stage: adding SAG, SHH and CHIR99021; third stage: adding SHH, SAG and FGF8b; fourth stage: adding SHH and FGF8b.
[0170] As in one embodiment of the present disclosure, the inventors also optimized the timing of addition of each supplementary component. Preferably, in multiple stages: first stage: culturing for 6~8 days from the beginning; preferably 7±0.5 days; second stage: culturing for 6~8 days to 11~13 days; preferably 12±0.5 days; third stage: culturing for 11~13 days to 18~20 days; preferably 19±0.5 days; fourth stage: culturing for 18~20 days to 31~33 days; preferably 32±0.5 days.
[0171] As in one embodiment of the present disclosure, the inventors also optimized the amount of each supplementary component. The optimization of added amounts is beneficial for obtaining/enriching specific midbrain substantia nigra dopaminergic nerve cells.
[0172] Midbrain substantia nigra dopaminergic nerve cells obtained by optimized method in the present disclosure exhibit excellent effects, including: repairing damaged neural circuits in brain or reconstructing neural functions; more specifically including: forming neural fibers in substantia nigra, projecting to the striatum to repair; or forming synaptic connections between neurons and target cells in brain to repair; projecting axons to the caudate putamen (CPu) to repair; forming neural fibers, along with the endogenous nigra-striatal neural connected pathway, growing specifically and extending to its endogenous target area-striatum to form neural connections with striatal neurons, and projecting to the striatum to repair.
Restorative Effects of Stem Cell-Derived Nerve Cells
[0173] Neurons are the basic functional unit of the brain. There are thousands of different types of neurons in brains of individuals. Complex and precise network connections (neural circuits) are formed between neurons, which is the basis for individuals to perceive the world, think and behave. Many neurological diseases, including stroke, cerebral trauma, and neurodegenerative diseases (Parkinson’s disease and Alzheimer’s disease, etc.), can lead to the loss of neurons and damage to neural connections in brain, resulting in severe neurological dysfunctions, such as hemiplegia, slow movements, muscle stiffness, impaired learning and memorable abilities, etc. However, the abilities of adult mammals, including humans, to regenerate nerves in brain are very limited. The loss of these neurons leads to the destruction of nerve connections and impaired nerve functions, with lack of effective treatments for diseases clinically. The key to stem cell therapy for neurological diseases is the restoration and functional reconstruction of damaged neural circuits. However, the precise network connections between neurons in brain of the individual are gradually formed during development, involving complex mechanisms of nerve fiber growth. In adult diseased brain environment, whether transplanted nerve cells can grow into nerve fibers, bridge “lost-connected” upstream and downstream brain regions, and repair damaged neural circuits still remains unclear. In one embodiment, is this restorative effect the result of random integration of transplanted cells or specific repair? What are the mechanisms and principles behind it? These are key issues to be solved urgently in the field of stem cell therapy for neurological diseases.
[0174] In view of the above problems, the inventors used Parkinson’s disease as a disease model to study the feasibilities and mechanisms of transplanting stem cell-derived nerve cells into the adult brain to repair damaged neural circuits. Parkinson’s disease is the world’s second largest neurodegenerative disease mainly manifested by static tremor, myotonia, bradykinesia, and so on. Progressive loss of dopaminergic neurons in the brain substantia nigra is the main reason, resulting in the destruction of the neural connections between the substantia nigra and the striatum, then causes insufficient dopamine secretion in the striatum, and eventually leads to motor deficits of patients. The inventors are committed to human stem cell neural differentiation technology for different types of neurons, and based on this, an efficient method for human stem cells to differentiate into midbrain substantia nigra dopaminergic neurons has been established. Furthermore, human stem cells were genetically marked by gene editing technology, and human dopaminergic neurons and their nerve fibers derived from stem cells could be specifically traced. The inventors transplanted genetically-marked human dopaminergic neurons into the damaged substantia nigra of Parkinson’s disease model mice, and found that the human dopaminergic neurons transplanted in the brain substantia nigra grew a large number of nerve fibers along with the endogenous nigra-striatum neural connected pathway, grew specifically and extended to its endogenous target area-striatum to form neural connections with striatal neurons, and most of the nerve fibers projected to the striatum. By genetic technology and rabies virus-mediated tracing technology, the inventors further traced the upstream neural innervation received by transplanted human dopaminergic neurons and found that transplanted human dopaminergic neurons received a similar neural innervation to endogenous substantia nigra dopamine. Studies on the electrophysiological function of neurons have found that transplanted human dopaminergic neurons exhibit similar electrophysiological characteristics to endogenous dopaminergic neurons in the substantia nigra of animals, regulated by similar neurotransmitters. These results indicated that human dopaminergic neurons transplanted into the brains of Parkinson’s disease model animals specifically repaired and reconstructed the damaged nigra-striatal neural connections, and their structures and functions were highly consistent with endogenous neural connections. Finally, by behavioral tests, the inventors found that the motor deficits of animals in the cell-transplanted group gradually improved with the prolongation of the transplantation. However, after inhibiting the activities of transplanted nerve cells by chemical genetics technology, the improvement of animal’s motor functions disappeared, suggesting that reconstructed neural functional connectivity by transplanted cells mediated the recovery of behavior in model animals. Interestingly, the inventors transplanted another type of nerve cells, human cortical glutamatergic neurons, into the substantia nigra of Parkinson’s disease model animals. The nerve fibers mainly projected to the cortex and olfactory bulb brain regions, with almost no projections in the striatum, which is hardly to repair damaged nigra-striatal neural circuit, also with motor deficits of model animals not be improved, indicating that only specific types of cells can repair specific neural functional circuits.
[0175] The present disclosure suggests that by transplanting nerve cells derived from stem cells, damaged nerve connections in the brain of adults can be repaired structurally and functionally and neural functions can be reconstructed. At the same time, the present disclosure also found that different types of nerve cells show different restorative effects on circuits, suggesting that for neurological diseases caused by the loss of different types of neurons, it is necessary to transplant specific nerve cells for circuit repair and treatment. These findings provide new ideas and theoretical basis for the treatment of brain injury and neurodegenerative diseases. Now the main types of nerve cells in the human brain can be efficiently obtained in vitro through stem cell neural differentiation technology. The development of stem cell technology will bring new hopes for the treatment of many neurological diseases.
[0176] Therefore, based on new findings embodiments of the present disclosure have the following characteristics: (1) the dopaminergic neurons derived from stem cells (such as human embryonic stem cells) have the characteristics of midbrain substantia nigra dopaminergic neurons; (2) functional input depends on the type of transplanted neurons rather than the location of transplantation; (3) midbrain dopaminergic neurons can precisely restore the nigra-striatal pathway; (4) functionally repaired nigra-striatal pathway restored motor functions in animal models of neurodegenerative diseases such as Parkinson’s disease.
Drug Screening
[0177] The inventors transplanted midbrain dopamine (mDA) or glutamate (Glu) cortical neurons derived from human embryonic stem cells (hESCs) into the substantia nigra or striatum of animal PD models, and found that the transplanted cells were widely integrated with the circuits in the host. Axonal pathways towards the dorsal striatum are determined by the type of transplanted neuron. Presynaptic inputs are largely dependent on the graft site, and inhibitory and excitatory inputs are determined by the type of transplanted neuron. hESC-derived mDA neurons exhibited characteristics of A9 neurons and restored the function of reconstituted nigra-striatal circuits to mediate improvements in motor functions. These results demonstrate similarities in cell-type-specific presynaptic and postsynaptic integrations between transplanted reconstructed circuits and endogenous neural networks, highlighting the abilities of hPSC-derived neuron subtypes in the adult brain for specific circuit repair and functional recovery.
[0178] Based on the new findings of the inventors, substances that repair damaged neural circuits in brain or remodel neural functions can be screened. Drugs useful for treating brain damage, neurodegenerative diseases, etc. can be found from the substances.
[0179] Therefore, the present disclosure provided a method for screening substances (including potential substances) for repairing damaged neural circuits or reconstructing neural functions, and the method includes: (1) treating a model system by a candidate substance, and the model system is neural circuit damaged or neural function damaged, and it includes dopaminergic neurons (dopaminergic nerve cells) in the midbrain; and (2) detecting the model system, if the candidate substance can statistically promote dopaminergic neurons to damaged neural circuits in the brain or promote their remodeling of neural functions, then the candidate substance is a useful substance for repairing damaged neural circuits or reconstructing neural functions.
[0180] Combined with research results of the present disclosure, after analyzing the effects of candidate substances on the repair of midbrain dopaminergic neurons for the nigra-striatal pathway, the pre-synaptic and post-synaptic integration of midbrain dopaminergic neurons, the ability of midbrain dopaminergic neuron axonal projections to dorsal regions and/or the athletic ability of animal models, the effectiveness of the candidate substances (potential substances or candidate drugs) can be determined.
[0181] Combined with research results of the present disclosure, by systematically analyzing the effects of candidate substances on the growth of midbrain dopaminergic neurons into nerve fibers, the specific growth and extension to its endogenous target area-striatumto form neural connections with striatal neurons and the projection to the striatum, the effectiveness of the candidate substances (potential substances or candidate drugs) can be determined.
[0182] In one embodiment of the disclosure, during screening, in order to more easily observe the changes before and after the treatment of candidate substance, a control group (Control) without adding the candidate substance (such as blank control or placebo control) can also be set.
[0183] On the other hand, the present disclosure also provided potential substances obtained by the screening method. These preliminary screening substances can constitute a library for screening, and people can finally screen for substances that can be really useful for repairing damaged neural circuits in brain or remodeling neural functions.
Pharmaceutical Preparation
[0184] The present disclosure also provided a pharmaceutical composition (preparation), and it includes effective amount (eg, 0.000001-50 wt%; preferably 0.00001-20 wt%; more preferably 0.0001-10 wt%) of midbrain substantia nigra dopaminergic nerve cells prepared by the method of the present disclosure, and a pharmaceutically acceptable carrier.
[0185] The term “pharmaceutically acceptable carrier” refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents. The term refers to a pharmaceutical carrier that is, by itself, not essential active ingredients and is not unduly toxic after administration. Suitable carriers may be used. Pharmaceutically acceptable carriers in compositions may include liquids such as water, saline, buffers. In addition, auxiliary substances such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. The carrier may also include cell transfection reagents.
[0186] The effective amount of midbrain substantia nigradopaminergic nerve cells of the present disclosure may vary with the form of administration, the severity of the disease to be treated, and the like. Selection of preferred effective amount can be determined based on various factors (e.g. through clinical trials). Such factors include but are not limited to: pharmacokinetic parameters, such as bioavailability, metabolism, half-life, the severity of the disease to be treated, weight, immune status of patients, the form of administration, and so on.
[0187] Specific therapeutically effective amount depends on a variety of factors, such as the particular condition to be treated, the physiological condition of the individual (e.g. the weight, age or sex), the type of individual being treated, the duration of the treatment, the concurrent treatments (if any) and specific formulation being used and the structure of the compound or its derivatives. For example, a therapeutically effective amount can be expressed as the total weight of active ingredients, such as in grams, milligrams, or micrograms; or as a ratio of the weight of active ingredients to body weight, such as in milligrams per kilogram of body weight (mg/kg). In one embodiment, an effective amount can be expressed as a concentration of an active ingredient (e.g. cells or cell preparations of the disclosure), such as molar concentration, weight concentration, volume concentration, molar weight concentration, mole fraction, weight fraction and mixing ratio. The human equivalent dose (HED) of an agent (such as the cells or cell preparations of the present disclosure) may be calculated based on the dose in animals. For example, estimates of the highest safe dose for human use based on “Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers” published by the Food and Drug Administration (FDA) may be made.
[0188] In a specific example of the present disclosure, some dosing regimens for animals such as mice are given. It is easy to convert the dosage of animals such as mice into dosages suitable for humans. For example, it can be calculated according to the Meeh-Rubner formula: A=k×(W.sup.⅔)/10,000. In the formula, A is the body surface area, calculated in m.sup.2 ; W is the body weight, calculated in g; K is a constant, which varies with animal species. Generally speaking, mice and rats are 9.1, guinea pigs are 9.8, rabbits are 10.1, cats are 9.9, dogs are 11.2, monkeys are 11.8, human is 10.6. It will be understood that, depending on the drug and the clinical situation, the conversion of the administered dose may vary according to the assessment of an experienced pharmacist.
[0189] The present disclosure also provides a kit including the pharmaceutical composition or directly including the midbrain substantia nigradopaminergic nerve cells. In addition, the kit may also includes instructions for using the medicine in the kit.
[0190] The disclosure if further illustrated by the specific examples described below. It should be understood that these examples are merely illustrative, and do not limit the scope of the present disclosure. The experimental methods without specifying the specific conditions in the following examples generally used the conventional conditions, such as those described in J. Sambrook, Molecular Cloning: A Laboratory Manual (3.sup.rd ed. Science Press, 2002) or followed the manufacturer’s recommendation.
Experimental Materials
Cell Culture
[0191] H9 hESCs and reporter H9 hESCs were cultured on a feeder of irradiated mouse embryonic fibroblasts (MEFs) in the ESC medium consisting of DMEM/F-12, KOSR, 1x NEAA, 0.5x Glutamax, 0.1 mM 2-Mercaptoethanol, and 4 ng/ml FGF-2. Cells were fed daily with fresh medium and passaged weekly by Dispase II.
Generation of Midbrain Dopamine Neurons and Forebrain Glutamate Neurons
[0192] Induction of the midbrain dopaminergic progenitors including: hESCs (1 day after passaging) on MEF feeder layer were cultured in the neural induction medium (NIM)(DMEM/F-12,1 x NEAA, 1× N2 supplement) supplemented with SB431542 (10 .Math.M) and DMH-1 (2 .Math.M). To pattern the differentiating cells to the midbrain FP progenitors, SHH (C2511, 500 ng/ml) and CHIR99021 (0.4 .Math.M) were added to the cultures from day 1 till day 7. On day 7, individual colonies of neuroepithelial cells were gently blown off with a pipette and replated on mouse embryonic fibroblast feeder in the NIM containing SAG (1 uM) and SHH (100 ng/ml) and CHIR99021 (0.4 uM) for additional 6 days (D7-12). On day 12, CHIR99021 was removed, SHH was reduced to 20 ng/ml, SAG (0.5 .Math.M) and FGF8b (100 ng/ml) was added to the culture to expand the progenitors in suspension till day 19. Then, 20 ng / ml SHH and 20 ng/ml FGF8b were kept in the neural induction medium till transplantation at day 32. For in vitro analysis, the neurospheres were dissociated by incubation in Accutase at 37° C. for 3-5 minutes on day 32 and plating onto glass coverslips that were coated with Matrigel. Cells were fed with Neural differentiation medium (Neurobasal Medium, 1×N2 supplement, 1×B27 supplement) (NDM) supplemented with brain-derived neurotrophic factor (BDNF, 10 ng/ml), glial cell line derived neurotrophic factor (GDNF, 10 ng/ml), ascorbic acid (AA, 200 uM), cAMP (1 uM), transforming growth factor β3 (1 ng/ml) and Compound E (0.1 .Math.M).
[0193] Induction of forebrain glutamate neurons from hESCs: H9 hESC colonies were cultured for 1 week with daily medium change. Then, hESC colonies were detached from the feeder layer and grown in the ES medium for 4 days to help form cell aggregates. For neural induction, cell aggregates were cultured in flasks fed with NIM supplemented with 2 .Math.M SB431542 and 2 .Math.M DMH-1 for 3 days. The ESC aggregates were then adhered to vitronectin coated 6-well plates in the presence of NIM till formation of neural tube-like rosettes at around day 16. The rosettes were gently blown off by a 1 mL pipette and suspended in the same medium for another 10 days. Then, progenitors were digested into small spheres with Accutase for 4 min at day 26. After an additional 1 day culture in flask with NIM, small spheres were collected and transplanted into animal models or seeded and matured in NDM supplemented with BDNF (10 ng/ml), GDNF(10 ng/ml), AA (200 .Math.M), cAMP (1 .Math.M),IGF1 (10 ng/ml) on glass coverslips for another 1 week for immunofluorescence staining.
PD Model and Cell Transplantation
[0194] The surgical procedure for producing the PD model was performed in SCID mice including: adult SCID mice (8-12 weeks) were anesthetized with 1%-2% isoflurane mixed in oxygen. 1 uL 6-OHDA (3 mg/ml, in saline with 1% ascorbic acid) was directly injected into the left substantial nigra (anterior-posterior [AP]=-2.9 mm, lateral [L]=-1.1 mm, vertical M= 4.5 mm, from skull). Animals with amphetamine-induced rotation at> 6/min over 1.5 h period were selected for cell transplantation 4 weeks after 6-OHDA-lesion surgery. Animals were randomly grouped and transplanted with glutamatergic progenitors, dopaminergic progenitors, or artificial cerebral spinal fluid (ACSF) (control). 50,000 cells were resuspended in 1 .Math.L ACSF containing Rock inhibitor (0.5 .Math.M),B27, 20 ng/ml BDNF and injected into the left nigra (anterior-posterior [AP]=-2.9 mm, lateral [L]=-1.1 mm, vertical [V] =4.4 mm, from skull) or left striatum (AP=+0.6 mm, L = 1.8 mm, V = 3.2 mm, from dura).
Donor Plasmid Construction
[0195] TALEN pair, Human codon-optimized Streptococcus pyogenes wild-type Cas9 (Cas9-2A-GFP), Cas9 nickase (Cas9D10A-2A-GFP), and pCAG-Flpo (plasmid #52342, plasmid #52341, plasmid #44719, plasmid #44720, plasmid #60662) was obtained from Addgene. PL652 donor plasmid vector containing an FRT-flanked PGK-puromycin expression cassette was constructed by replacing the loxP sequence in the PL552 (#68407) with the FRT sequence.
[0196] To generate the TH-iCre donor plasmid, DNA fragments with left or right homology arm were PCR amplified from the genomic DNA immediately upstream or downstream of the STOP codon of TH gene. The DNA fragment with iCre gene fused to P2A sequence was PCR amplified from pDIRE (Addgene plasmid #80945). P2A sequences were included in the PCR primers. These three fragments were then cloned into the multiple cloning sites of plasmid PL652.
[0197] To generate TH-tdTomato donor plasmid, the DNA fragment with tdTomato gene fused to P2A was PCR amplified from pAAV-FLEX-ArchT-tdTomato (Addgene plasmid #28305). P2A sequences were included in the PCR primers. The DNA fragment with hGH polyA signal sequences was PCR amplified from AAVS1-pur-CAG-EGFP plasmid (Addgene plasmid #80945). The DNA fragments with left or right homology arm for TH-iCre donor plasmid, DNA fragment containing P2A-tdTomato sequences, and DNA fragments containing hGH poly A signal sequences were cloned into the multiple cloning sites of plasmid PL552.
[0198] To generate AAVS1-neo-CAG-ChR2-EYFP donor plasmid, the inventors replaced the FlpeERT2 gene in the AAVS1-Neo-CAG-Flpe-ERT2 plasmid (Addgene plasmid #68460) with ChR2-EYFP gene which is PCR amplified from pAAV-hSyn-hChR2 (H134R)-EYFP (Addgene plasmid #26973).
[0199] To generate the AAVS1-pur-CAG-Bi-DREADD donor plasmid (AAVS1-pur-CAG-hM3Dq-mcherry-P2A-HA-KORD) or AAVS1-pur-CAG-mCherry donor plasmid, the inventor amplified mCherry or hM3Dq-mCherry from AAVS1-pur-CAG-hM3Dq-mCherry (Addgene plasmidl #80948), HA-KORD from pAAV-hSyn-dF-HA-KORD-IRES-mCitrine (Addgene plasmid #65417). The two DREADD genes, hMsDq-mcherry and HA-KORD, were linked by P2A peptide (hM3Dq-mcherry-P2A-HA-KORD) to ensure simultaneous expression of these two genes in the same cells. The hMaDq-mcherry-P2A-HA-KORD or mCherry gene was inserted into the AAVS1-pur-CAG-EGFP to replace EGFP. SA-Neo, splice acceptor sequence followed by T2A self-cleaving peptide sequence, and then the neomycin resistance gene. CAG, synthetic CAGGS promoter with the actin enhancer and the cytomegalo-virus early promoter.
Electroporation and Generation of Reporter hESC Lines
[0200] H9 hESCs were pretreated with Rho-kinase (ROCK) inhibitor for 6-8 h (0.5 mM). Cells were then digested by TrypLE™ Express Enzyme, dispersed into single cells, and electroporated with appropriate plasmids in 500 mL of electroporation buffer (5 mM KCI, 5 mM MgCI.sub.2, 15 mM HEPES, 102.94 mM Na.sub.2HPO.sub.4, and 47.06 mM NaH.sub.2PO.sub.4, pH=7.2) using the Gene Pulser Xcell System (Bio-Rad) at 250 V, 500 mF in a 0.4 cm cuvette (Phenix Research Products). Cells were then seeded on MEF feeder layer in 6-well plate in MEF-conditioned medium with ROCK inhibitor. Cells were fed daily with MEF-conditioned ESC medium (CM). 72 hours later, puromycin (0.5 .Math.g/ml) or G418 (50-100 .Math.g/ml) were added into CM for selection for two weeks. After selection, cells were pretreated with ROCK inhibitor for 6-8 h, and then individual clones were picked up. Genomic PCR was applied to examine the integration of the transgene.
[0201] For generation of TH-iCre knock-in hESC line, the cassette containing a P2A peptide sequence-linked codon-improved Cre recombinase (iCre) gene with a STOP codon and then FRT-flanked PGK-Puro sequence (PGK promoter-driven) was introduced to immediately upstream of the STOP codon of the endogenous TH gene of H9 ESCs. FRT-flanked PGK-Pur was then removed by transient expression of Flpo.
[0202] For generation of TH-tdTomato/AAVS1-ChR2-EYFP hESC line, the cassette containing a P2A peptide sequence-linked tdTomato gene with STOP codon followed by polyA sequence and then PGK-Pur sequence was introduced to immediate upstream of the STOP codon of the endogenous TH gene of H9 ESCs by CRISPR, and then the ChR2 expression cassette was knocked into the AAVS1 locus by TALEN. For generation of Bi-DREADD or mCherry hESCs, the hMDq-mCherry-P2A-HA-KORD or mCherry expression cassette was inserted into the AAVS1 locus of H9 hESCs using TALEN.
Whole-Cell Patch-Clamp and Brain Slice Recording
[0203] Coronal brain slices (350 um thick) at the level of the forebrain or the midbrain were prepared from recovered animals at 3 and 6 months post-transplantation using a vibratome (Leica VT1200S) in ice-cold cutting solution (100 mM glucose, 75 mM NaCI, 26 mM NaHCO3, 2.5 mM KCI, 2 mM MgCI2-6H2O, 1.25 mM NaH2PO4-6H2O, and 0.7 mM CaCI2). The slices were transferred to the recording artificial cerebrospinal fluid (ACSF, 124 mM NaCI, 4.4 mM KCI, 2 mM CaCI2, 1 mM MgSO4, 25 mM NaHCO3, 1 mM NaH2P04, and 10 mM glucose) saturated with 95% 02/5% CO2. Voltage and current signals were recorded by Axon 700B amplifier (Axon). The recording electrodes (3-5 MΩ) were filled with a solution containing 112 mM Cs-Gluconate, 5 mM TEA-CI, 3.7 mM NaCI, 0.2 mM EGTA, 10 mM HEPES, 2 mM MgATP, 0.3 mM Na3GTP and 5 mM QX-314 (adjusted to PH 7.2 with CsOH) for spontaneous excitatory post-synaptic current (sEPSC) and spontaneous inhibitory post-synaptic current (sIPSC) recording. For sEPSC or sIPSC recording, cells were voltage clamped at 60 mV or 0 mV, respectively. The initial access resistance was monitored throughout the experiment, ranging from 15-30 MΩ. Cells with the access resistance changed > 15% were discarded. Data were filtered at 1 kHz and digitized at 10 kHz. Action potentials (APs) in response to the blue light stimulation (473 nm, frequency 5 Hz, intensity 10 mM/mm2) or the depolarizing currents (0-100 pA, step 10 pA, duration 2 s) were recorded in current clamp mood. Sag measurement was conducted under current clamp mode by injection of 90 pA or 120 pA currents into the grafted or endogenous mDA neurons, respectively. Transplanted mDA neurons and non-mDA neurons were identified by their tdTomato fluorescence in the EYFP-positive graft.
Viral Injections and Rabies Tracing Experiments
[0204] For rabies tracing experiments, 200 nL AAV expressing Cre-dependent TVA and tdTomato (AAV2 / 9-Efla-DIO-TVA-2A-NLS-tdTomato, titer 1.29*10^12 genome copies (gc)/ ml ), or 200 nL AAV expressing Cre-dependent Rabies Glycoprotein (AAV2 / 9-Efla-DIO-G, titer 1.29*10^12gc/ ml) were co-injected into the graft site (For nigral graft: AP=-2.9 mm, L=1.1 mm, V=4.4 mm, from skull; For striatum graft: AP= +0.6 mm, L= -1.8 mm, V= 3.2 mm, from dura) of PD mice 5 months after transplantation. 3 weeks later, EnVA-pseudotyped, rabies G deleted, EGFP-expressing rabies virus (RVdG-EGFP, 400 nl, titer 2*10^8pfu /ml) was injected into the same site for trans-synaptic labeling. One week later, the mice were sacrificed for histological analysis. For endogenous mDA neuron, viruses were injected to the SNc (AP= 2.9 mm, L= 1.1 mm, V= 4.5 mm, from skull) of DAT-Cre / Ai9 mice. After fixation, the brain was sectioned (30 um thick) with a freezing microtome. All coronal sections (1:4 series) without staining were imaged by a 20x objective with a fluorescence microscope (Olympus VS120). Tiled images were automatically stitched using a 10% overlap with VS-ASW (Olympus) software. The locations of labeled neurons and the outlines of brain areas were manually labeled using Photoshop according to Paxinos and Franklin (2007). Some sections underwent immunostaining to elucidate cell identity.
Tissue Preparation and Immunohistochemistry
[0205] Animals were sacrificed with an overdose of pentobarbital (250 mg/kg, i.p.) and perfused with saline followed by 4% ice-cold phos-1 phate-buffered paraformaldehyde (PFA). The brains were removed and immersed sequentially in 20% and 30% sucrose until sunk. Serial sagittal (0.12 to 3.12 mm from medial to lateral) or coronal (1.42 to 0.10 mm from the Bregma) sections were cut on a freezing microtome (Leica SM2010R) at a thickness of 30 mm and stored at 20° C. in a cryoprotectant solution. Free-floating sections were incubated with a primary antibody in 4° C. for 1-2 nights, and then the unbound primary antibodies were removed. For DAB staining, sections were incubated with corresponding biotinylated secondary antibodies for 1h followed by avidin-biotin peroxidase for 1h at room temperature. Immunoreactivity was visualized with DAB staining kit. The sections were then dehydrated with ethanol, permeabilized in xylene, and mounted in neutral resin. For fluorescent immunolabeling, sections were incubated with corresponding fluorescent secondary antibodies for 1 h at room temperature. Then sections were mounted by Fluoromount-G.
Packaging of Lentivirus
[0206] Lentiviruses were generated in 293T cells by transfecting packaging and backbone plasmids using calcium phosphate/DNA copre-cipitation method. 293T cell were cultured in Dulbecco’s MEM (DMEM) containing 10% FBS. The supernatant containing the viral particles was collected 72 hours after transfection, and concentrated by ultracentrifugation at 27000 rpm for 2 hours at 4° C. The viral particles were then resuspended in DPBS.
Imaging and Cellular Quantification
[0207] To quantify the population of EN1, FOXA2, LMX1A, NURR1, GIRK2, and TUJ-1 expressing cells among total TH or TH to total cells, at least five randomly chosen images from coverslips were counted with ImageJ software. Data were replicated three times and were expressed as mean ± SEM. For measuring the human fiber density in the brain slices, tiled images were captured by a Nikon TE600 or Olympus VS 120 microscope. The optical density of human fibers in different areas of the mouse brain was measured by image processing and analysis system (Image Pro Plus 5.1 software). Data were shown as optical density in different areas. For TH, GIRK2, LMX1A, human nuclei (hN) and FOXA2 staining, the graft was outlined and captured by a 60x objective with Nikon A1R-Si laser-scanning confocal microscope (Nikon, Tokyo, Japan) or a fluorescence microscope (Olympus VS120). Single or double stained cells were counted manually with ImageJ. Data were presented as ratio of TH-, LMX1A-, FOXA2- to total hN or GIRK2/TH/hN to TH/hN cells. All data are expressed as mean ± SEM.
Behavioral Test: Rotation Test
[0208] Amphetamine-induced rotations were tested before transplantation and every month till 6 months after transplantation. Rotation was recorded by a video camera for 1.5 h, 5-10 min after peritoneally amphetamine (2 mg/ml in saline, 5 mg/kg) injection. Data were presented as the average net number of rotations per minute during 90 min.
[0209] For spontaneous rotation test, animals were recorded for 60 min after injection of CNO (1.2 mg/kg) for 20 min, SALB (5 mg/kg) for 5 min, or saline for 20 min.
Behavioral Test: Cylinder Test
[0210] Individual animal was placed in a glass cylinder and recorded by a camera for 3 min. The ipsilateral and contralateral paw touches to the wall of the cylinder were counted. The data were expressed as the percentage of ipsilateral touches to total touches. For drug treatment, animals were treated by CNO (1.2 mg/kg) for 20 min, SALB (5 mg/kg) for 5 min, or saline for 20 min before Cylinder test.
Behavioral Test: Rotarod Test
[0211] An accelerating Rotarod (Med Associates Instruments) was used to test motor coordination. All animals were pre-trained for two days in order to reach a stable performance. On day 1, mice were trained on a rotating rod that accelerated from 2 per minute (rpm) to 20 rpm in a period of 300 s for three times. On day 2, mice were trained on rod accelerated from 3 rpm to 30 rpm twice, and from 4 rpm to 40 rpm once, in a period of 300 s. The test was performed from the third day on a rotating rod that accelerated from 4 rpm to 40 rpm in a period of 300 s. The period of time the mouse stayed on the rod was monitored. The average duration from three repeated tests of each animal was used for data analysis.
Quantification and Statistical Analysis
[0212] SPSS software was used for statistical analysis. In all studies, data were analyzed by Student-t test, Paired t test, two-way ANOVA followed by Holm-Sidak test, Two-way RM ANOVA followed by Tukey’s post hoc test, or One-way ANOVA followed by Holm-Sidak test. Statistical significance was determined at p < 0.05.
[0213] English notes of all abbreviation in the present disclosure are shown in Table 1.
TABLE-US-00001 Acb Accumbens nucleus Pa Paraventricular hypothalamic nucleus AcbC Accumbens nucleus core PAG Periaqueductal gray AcbSh Accumbens nucleus shell PB Parabrachial nucleus AI Agranular insular cortex PF Paratascicular thalamic nucleus Amy Amygdala PLH Peduncular part of lateral hypothalamus AOV Anterior olfactory nucleus, ventral part Pn Pourinereticular nucleus APT Anterior pretectal nucleus PO Preoptic area ASt Amygdala strianal transition area PrCnF Precuneiform area Ce Centralnucleus of the amygdala SI Substantia innorminara CPu Candate puiamen SN Substantia nigra DR Dorsal raphe nucleus SNC Substantia nigra, pars compacta DTT Dorsal tenia tecta SNR Substantia nigra, pars reticularis EA Extended amygdala ST Bed nucleus of the stria terminalis FrA Frontal assosiation cortex G Geniculnte nucleus STh Subthalmnic nucleus GP Globus pallidus Tu Olfactory tubercle Hipp Hippocampus VM Ventromedial thalamic nucleus HT Hypothakumus VMH Ventromedial hypothalamic nucleus IPAC Interstitial nucleus of the posterior limb of the anterior VP Ventral palllidum LPO Lateral preoptic nucleus VS Ventral strianum M Motor cortex ZI Zona incerta M1 Primary motor cortex M2 Secondary cortex MD Mediodorsalthalamic nucleus MFB Medial forebrain bundle MnR Median raphe nucleus MPA Medial preceptic area mRt Mesancephalic reticular formation OB Olfactory bulb
Example 1. Grafted Human mDA and Glu Neurons Project to Differential Targets
[0214] During development, targeted axonal projection is often determined by the intrinsic properties of the cells. To address whether cell-intrinsic properties also determine target finding in the adult brain, the inventors transplanted hESC-derived mDA or forebrain Glu neuron progenitors into the midbrain of PD model mice. mDA or Glu neuron progenitors were differentiated from hESCs according to the protocols of the inventors. On day 32 of mDA neuron differentiation (the day of transplantation), most of the progenitors expressed the floor plate and midbrain markers CORIN, FOXA2, LMX1A, and EN1 (
[0215] The inventors then transplanted the mDA (
[0216] In contrast, Glu neurons extended axons locally, filling the entire midbrain (
[0217] Together, these results indicate that the grafted human neural progenitors differentiate to respective neuronal types and project axons to different brain regions.
Example 2. Grafted Human mDA Neurons Project Through Their Cognate Path
[0218] Examination from serial sagittal sections (
[0219] In the dorsal/lateral striatum, the dense hNCAM fibers exhibited elaborately ramified, fine-beaded terminal networks (
[0220] Immunohistochemistry analysis of the mDA graft showed that 68% of the grafted cells co-expressed TH and human nuclei (hNs), and most of the TH+ cells also expressed GIRK2 as well as FOXA2 and LMX1A (
Example 3. Genetic Labeling Reveals Specific Axonal Innervation by Human mDA Neurons
[0221] To elucidate the specific axonal innervation by grafted mDA neurons, we created a TH reporter hESC line with tdTomato expression recapitulating that of the endogenous TH gene (Method Details). The inventors further knocked in a ChR2-EYFP fusion protein expression cassette into the AAVS1 locus to enable specific labeling and manipulation of transplanted human cells (
[0222] We then transplanted the mDA progenitors derived from TH-tdTomato/AAVS1-ChR2-EYFP hESCs into the SN or striatum of PD model mice (
[0223] The cellular features (
[0224] Together, these results indicate that grafted human mDA neurons have functional characteristics of A9 mDA neurons.
Example 4. Anatomical Synaptic Inputs to Human Neurons Are Associated with Transplant Sites
[0225] Rabies-mediated tracing has been used to track anatomical inputs onto transplanted cells in the PD model. To reveal presynaptic inputs specifically to grafted human mDA neurons, the inventors combined the Cre-loxP gene expression system with rabies-mediated transsynaptic tracing (
[0226] The starter neurons (EGFP+/tdTomato+) were only found in human grafts, and they were TH+ (
[0227] In the brain with the striatal graft, dense presynaptic inputs were found in the CPu but not the Acb. More inputs were observed in the GP and cortical areas compared with those to nigrally grafted human mDA neurons. The parafascicular thalamic nucleus (PF) and mediodorsal thalamic nucleus (MD) preferentially projected to striatally grafted human mDA neurons. Labeled host neurons were also found in the Ce and SN reticular part (SNR) in the brain with a striatal graft. Few labeled neuronswere found in the more caudal brain regions, such as the PAG, DR, parabrachial nucleus (PB), and Pn (
[0228] Thus, human mDA neurons grafted in the striatum and nigra receive inputs from largely different brain regions, suggesting location-dependent presynaptic inputs. Nigrally grafted human mDA neurons receive extensive inputs from similar brain regions, as endogenous mDA neurons do.
Example 5. The Identity of a Grafted Neuron Determines Its Functional Input Characteristics
[0229] Electrophysiological recording showed that few sEPSCs and sIPSCs (spontaneous excitatory and inhibitory postsynaptic currents, respectively) were detected in human mDA and non-mDA neurons in striatal or nigral grafts 3 months after transplantation (
[0230] Interestingly, the sIPSC frequency in mDA neurons was higher than that in non-mDA neurons in the nigral grafts (
[0231] These results suggest that the identity of the grafted neurons, rather than the graft site, determines the inhibitory and excitatory input characteristics of the grafted neurons.
Example 6. Transplantation of mDA but Not Glu Neurons Corrects Motor Deficits in PD Mice
[0232] The functional effect of the nigral grafts was assessed by amphetamine-induced rotation, rotarod test, and cylinder test before and every 4 weeks after grafting (
[0233] In the rotarod test, which is used to assess motor coordination and balance and does not depend on pharmacological stimulation of the dopaminergic system, the latency to fall was significantly increased over time in PD mice that received nigral orstriatal transplantation of human mDA neurons (p < 0.001) but not in those that received nigral Glu neurons or ACSF (p >0.05;
[0234] In the cylinder test, a measure of forelimb akinesia, all groups presented preferential ipsilateral touches after 6-OHDA lesion. The ipsilateral touching preferences were reduced significantly (close to 50%) 4 months after nigral or striatal transplantation of human mDA-rich neurons (p < 0.001) but not nigral Glu neurons or ACSF (p> 0.05;
Example 7. Motor Recovery Depends on Functional Reconstruction of the Nigra-Striatal Circuit
[0235] To determine whether behavioral recovery of PD-model animals depends on the reconstructed nigra-striatal circuit, we transplanted mDA neurons that were derived from hESCs with a “bi-directional switch″(
[0236] Using the cylinder test and spontaneous rotation test, which do not require stimulation of DA release by amphetamine, we found that CNO (1 mg/kg) treatment further decreased the preferential ipsilateral touches (p <0.05;
[0237] These results suggest that recovery of forelimb akinesia and asymmetric rotation depends on graft activity.
Discussion
[0238] This disclosure developed genetic labeling strategies to precisely map the projection from and synaptic inputs to grafted human mDA neurons in a mouse model of PD. The inventors found that human mDA neurons transplanted into the nigra specifically projected to the dorsal striatum. The grafted mDA neurons receive synaptic inputs, as revealed by rabies-mediated tracing, in a pattern strikingly similar to those to endogenous mDA neurons. Electrophysiological recording revealed predominantly inhibitory inputs to the grafted mDA neurons, which appears to be dependent on cell identity but not transplant site. With pre- and post-synaptic integration, homotopically grafted human mDA neurons rescue the motor deficits of PD mice in a manner dependent on graft activity. These findings reveal cell-type-dependent functional circuit integration by transplanted neurons, highlighting the prospect of using specialized neuronal types from stem cells to repair the neural circuit to treat neurological conditions.
[0239] What determines the targeted projection by grafted neurons in the mature brain remains unknown. By transplanting two types of projection neurons, mDA and Glu neurons, into the SN of PD mice, the inventors found that both neuronal types project axons over a long distance, but to different targets, via distinct routes, with the majority of axons of grafted mDA neurons targeting the CPu. This is further verified by using TH reporter cells, showing nearly exclusive projection to the dorsal (CPu) but not ventral striatum (Acb). Because the CPu is the main target of SNc (A9) mDA neurons (Björklund and Dunnett, 2007; Joel and Weiner, 2000), the inventors’ finding suggests that human mDA neurons are mostly A9-like cells. Indeed, cellular characterization of the inventors and, in particular, electrophysiological recording confirm an A9 identity of the human mDA neurons. This interpretation suggests that many of the axonal projections to other brain regions seen in the previous studies may be coming from non-mDA neurons. Together, these results strongly suggest that pathfinding and target projection are largely determined by the identity of the grafted neurons.
[0240] Correct synaptic inputs into grafted neurons are also critical for restoration of lost function. In the present disclosure, the TH-iCre system specifically enables identification of monosynaptic inputs to grafted human mDA neurons, revealing an interesting pattern. By looking at the graft in general, the anatomical synaptic inputs seem to be associated with the graft sites even though nigrally and striatally grafted mDA neurons receive inputs from overlapping areas, like the dorsal striatum, similar to the observation made by Adler et al. However, the striking similarity of the inputs to nigrally grafted human mDA neurons and endogenous mDA neurons suggests that cell identity plays a role in dictating synaptic inputs. This is displayed more clearly at the functional level. Grafted mDA neurons receive more inhibitory but less excitatory inputs compared with non-mDA neurons, regardless of whether the cells are transplanted into the striatumor nigra, suggesting that the identity of the grafted neurons dictates the functional synaptic inputs.
[0241] With pre- and post-synaptic integration by transplanted mDA neurons, it is natural to believe that the reconstructed nigra-striatal circuit contributes to motor recovery of PD mice. It has been shown that human mDA neurons transplanted into the striatum functionally connect with striatal neurons and contribute to animal behavior recovery using optogenetic and chemogenetic tools. In the current study, use of the Bi-DREADD strategy, which enables excitation and inhibition on the same grafted cells, clearly demonstrates that the reconstructed nigra-striatal circuit is functional, underlying the behavior recovery of PD mice.
[0242] Taken together with precise genetic labeling and functional measurements, we revealed that restoration of a neural circuit by transplanted cells in the mature brain, including pathfinding, targeting specificity, and functional input establishment, is largely determined by the intrinsic properties of the grafted neurons. Therefore, it is critical to transplant highly enriched, appropriately fated neural progenitors to achieve reconstruction of specific circuits for therapeutic outcomes. Hence, cell-based therapy to treat neurological conditions is realistic.
[0243] Each reference provided herein is incorporated by reference to the same extent as if each reference was individually incorporated by reference. In addition, it should be understood that based on the above teaching content of the disclosure, and various changes or modifications to the disclosure may be made, and these equivalent forms also fall within the scope of the appended claims.